Figure 5.

HuR upregulates cell surface distribution of TLR2 via CNPY3. (A) Interaction of CNPY3 with TLR2 in normal IECs as measured by immunoprecipitation (IP) assays using anti-TLR2 antibody. (B) Immunoblots of TLR2 and CNPY3 in the IP materials using anti-DDK antibody in cells transfected with a expression vector encoding DDK-taged CNPY3. (C) Immunoblots of membrane (Mem) and cytoplasimc (Cyto) TLR2 in CNPY3-silencent cells: i) total proteins; and ii) subcellular distribution of TLR2. Whole-cell lysates were harvested 48 h after transfection with siRNA targetting CNPY3 (siCNPY3) or C-siRNA. Membrane and cytoplasmic proteins were isolated, and equal loading was monitored by coomassie staining. (D) Quantitative analysis derived from densitometric scans of immunoblots of TLR2 in cells described in C (n = 3). (E) Western blot analysis of the abundance of HuR, CNPY3, and TLR2 in cells transfected with siHuR or cotransfected with siHuR and a CNPY3 expression vector for 48 h. (F) Immunoblots of membrane and cytoplasmic TLR2 in cells described in (E). (G) Quantitative analysis derived from densitometric scans of immunoblots of TLR2 in cells described in F (n = 3). (H) Activity of luciferase (Luc) NF-κB reporter gene in HuR-silenced cells (siHuR) transfected with the CNPY3 expression vector (n = 3). Twnty-four hours after transfection with the Luc-NF-κB reporter, cells were exposed to the TLR2 ligand, Pam3csk4 (10 ng/ml). The Luc activity was examined 6 h after treatment owth Pam3csk4. *P < 0.05 compared with solvent; ⁺P < 0.05 compared with cells transfected with C-siRNA or cotransfected with siHuR and CNPY3 and then treated with Pam3csk4.